GSM5751568: M4; Mus musculus; RNA-Seq
Liver non-parenchymal cells were isolated in a two-step perfusion procedure. The hepatocytes were removed by centrifugation and F4/80+ cells were isolated from non-parenchymal cells using Anti-F4/80 MicroBeads (130-110-443, Miltenyi Biotec) according to the protocol supplied by the manufacturer. High-quality RNA was prepared from F4/80+ non-parenchymal cells using a combined TRIzol and RNeasy Mini Kit (Qiagen) method. RNA quality control was performed using the Nanodrop Spectrophotometer (Thermo Fisher) and TapeStation System (Agilent). 250~300bp insert cDNA library preparation was carried out by Novogene. Generally mRNA was enriched using oligo(dT) beads. mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina), dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment. (Method provided by Novogene)